The long-range goal of these studies is to elucidate the mechanism(s) of bone, dentin, and cementum mineralization and thereby provide new avenues for therapy for those dental and orthopaedic conditions where mineralization is impaired or excessive. Animals and humans with mutated or ablated genes coding for major extracellular matrix (ECM) proteins display abnormal mineral in teeth and bones, implying that ECM proteins are involved in the formation of healthy, physiologically sound mineralized tissues. Results obtained during the past funding period indicate that ECM proteins can regulate both the formation and proliferation of physiologic hydroxyapatite (HA) mineral crystals in a collagenous matrix by interacting with nascent, newly formed, and growing crystal surfaces. This interaction is dependent on protein concentration, post-translational modification, and interaction with other ECM components (including collagen). Based on these observations, we hypothesize that 1) Post translational modifications are essential for the activity of ECM proteins involved in mineralization. In particular, a) although phosphorylation is essential not all phosphorylations are equally important, and b) fragmentation of ECM proteins yields active products that have different effects on mineralization than intact protein. 2) Binding of ECM proteins to fibrillar collagen alters their ability to regulate mineralization. 3) When ECM proteins active in the mineralization process bind to collagen, other proteins, HA, and/or Ca+2 they undergo a conformational change that may regulate the mineralization process. There are 3 specific aims that use similar methodologies to test these hypotheses: Aim 1: To study the effects of variously processed forms of 3 SIBLING proteins on HA formation and growth in the presence and absence of fibrillar collagen: osteopontin (OPN) fragments, matrix extracellular phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (dssp). For each of these proteins we will probe mechanism of action by characterizing HA formation in a cell-free in vitro system and determining secondary structures of proteins that have statistically significant effects on HA formation and growth. We will determine whether differences observed in such solution studies are due to variations in phosphorylation, to binding to HA or fibrillar collagen, and/or to conformational changes. We will also do in situ analyses using a combination of EM, micro-CT, and FTIR-imaging to characterize the mineral phenotype in teeth and bones of knockout (KO) and overexpression (TG) animals and develop working models to explain these phenotypes based on mechanism(s) postulated from the in vitro studies. Aim 2: To determine the effects of the small leucine-rich proteoglycans (SLRPs), in the presence and absence of fibrillar collagen, on HA formation and growth in cell-free in vitro and in situ systems. Aim 3: To determine whether DSPP and BGN interact synergistically during the collagen-based in vitro mineralization process using in vitro and in situ analyses.